Rett syndrome (RTT) is a devastating neurodevelopmental disorder caused by loss-of-function mutations in the X-linked MECP2 gene. MECP2 encodes methyl-CpG-binding protein 2 (MeCP2), an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. How MeCP2 deficiency causes neurological deficits remains poorly understood, but it is clearly related to dendritic and synaptic abnormalities. We previously reported that MeCP2-deficient microglia (MDM) cause excitotoxicity by constitutively releasing five times more glutamate than wild-type microglia, thus damaging dendrites and synapses. We subsequently found that MeCP2 is a potent transcriptional suppressor of the glutamine transporters SNAT1 and SNAT2. MDM consequently show over-expression of SNAT1 and SNAT2, resulting in increased glutamine uptake, disruption of microglial glutamine homeostasis, mitochondrial oxidative stress, and over-production of glutamate. This novel MeCP2-regulated pathway is highly significant for identification of therapeutic targets to block microglial neurotoxicity in RTT. Because our studies reveal that microglial glutamate production is regulated by a major epigenetic factor MeCP2, the research into this pathway will advance our knowledge about how neural activities regulate heterogeneous microglia response patterns and neuron-glia interactions through the epigenetic interface of DNA methylation. To consolidate the support for this mechanism, the purpose of this proposal, therefore, is (1) to firmly establish the link between MeCP2-regulated SNAT1/SNAT2 expression and MDM abnormalities and (2) to examine the relevance of this pathway to RTT. In Aim 1, we will determine the causal relationship between SNAT1/SNAT2 expression and the MDM phenotype, such as mitochondrial abnormalities and glutamate over- production. We will determine if over-expression of SNAT1 and/or SNAT2 in wild-type microglia induces the MDM phenotype. We will also determine if knock-down of SNAT1 and/or SNAT2 expression in MDM reduces the MDM phenotype. In Aim 2, we will determine the progression of microglial abnormalities (SNAT1/SNAT2 expression/activity and mitochondrial abnormalities) at different disease stages, using microglia freshly isolated from brains of MECP2 knockout mice and control wild-type mice. We recently have established a series of techniques to study microglia freshly isolated from juvenile and mature rodent brains, which would best represent microglia in vivo. We will employ qRT-PCR, flow cytometry, patch-clamp, electron microscopy, and fluorescent imaging to study the MDM phenotype in this preparation. The results of these studies are expected to be highly significant by 1) helping us to understand how MeCP2 deficiency causes dendritic and synaptic abnormalities in RTT through a microglia-mediated mechanism; 2) exploring the role of glutamine transporters in microglial pathology, which is presently poorly understood; and 3) providing one of the first few studies to examine epigenetic control of microglia function.